1. Field of the Invention
The present invention relates to a non-desensitizing mutant of the transient receptor potential TRPM5 ion channel. The present invention also relates to a high throughput screening method useful in the identification of compounds that affect taste sensation by modulating the activity of the non-desensitizing mutant TRPM5 ion channel. The screening method, using for example, electrophysiological measurements, allows for rapidly screening compounds during the non-desensitizing TRPM5 ion channel's prolonged activation period. The non-desensitizing mutant TRPM5 ion channel also allows TRPM5 activation to be monitored with assay techniques that require a longer period of ion channel activation. Therefore, the non-desensitizing mutant TRPM5 ion channel does not limit the types of high throughput screening methods that can be employed by its duration of activation.
2. Background
Taste perception not only plays a critical role in the nutritional status of human beings, but is also essential for the survival of both lower and higher animals (Margolskee, R. F. J. Biol. Chem. 277:1-4 (2002); Avenet, P. and Lindemann, B. J. Membrane Biol. 112:1-8 (1989)). Taste perception is carried out by taste receptor cells (TRCs). TRCs perceive the multitude of compounds that are associated with a given taste, and convert that perception to a signal deciphered by the brain, resulting in sweet, bitter, sour, salty, or umami (savory) taste.
TRCs are polarized epithelial cells, meaning they have specialized apical and basolateral membranes. Taste buds contain 60-100 TRCs, each having a tiny portion of its membrane exposed on the mucosal surface of the tongue (Kinnamon, S. C. TINS 11:491-496 (1988)). Sensory transduction is initiated by sapid molecules, or “tastants,” that interact with microvillar processes on the apical membrane of TRCs. The tastants bind specific membrane receptors, leading to a voltage change across the cell membrane; in turn this depolarizes, or changes the electric potential of the cell, causing transmitter release and excitation of primary gustatory nerve fibers.
Ion channels are transmembrane proteins that form pores in a membrane and allow ions to pass from one side to the other (reviewed in B. Hille (Ed), 1992, Ionic Channels of Excitable Membranes 2nd ed., Sinauer, Sunderland, Mass.). Although certain ion channels are open under all physiological membrane conditions (so-called leaky channels), many channels have “gates” that open in response to a specific stimulus. As examples, voltage-gated channels respond to a change in the electric potential across the membrane, mechanically-gated channels respond to mechanical stimulation of the membrane, and ligand-gated channels respond to the binding of specific molecules. Various ligand-gated channels can open in response to extracellular factors, such as neurotransmitters (transmitter-gated channels), or intracellular factors, such as ions (ion-gated channels), or nucleotides (nucleotide-gated channels). Still other ion channels are modulated by interactions with proteins, such as G-proteins (G-protein coupled receptors or GPCRs).
One recently discovered ion channel, TRPM5, has been shown to be essential for taste transduction. Perez et al., Nature Neuroscience 5:1169-1176 (2002); Zhang et al., Cell 112:293-301 (2003). TRPM5 is a member of the transient receptor potential (TRP) family of ion channels. TRPM5 forms a channel through the membrane of the taste receptor cell, and is believed to be activated by stimulation of a receptor pathway coupled to phospholipase C and by IP3-mediated Ca2+ release. The opening of this channel is dependent on a rise in intracellular Ca2+ levels. Hofmann et al., Current Biol. 13:1153-1158 (2003). The activation of this channel leads to depolarization of the TRC, which in turn leads to transmitter release and excitation of primary gustatory nerve fibers.
Because TRPM5 is a necessary part of the taste-perception machinery, its inhibition prevents an animal from sensing tastes. Although taste perception is a vital function, the inhibition, or masking, of undesirable tastes is beneficial under certain circumstances. For example, many active pharmaceutical ingredients of medicines produce undesirable tastes, such as a bitter taste. Inhibition of the bitter taste produced by the medicine may lead to improved acceptance by the patient. In other circumstances, enhancement of taste may be desirable as in the case of developing improved artificial sweeteners or in treatment of taste losses in groups such as the elderly. Mojet et al., Chem. Senses 26:845-60 (2001).
TRPM5 displays voltage modulation and rapid activation/deactivation (“opening and closing”) kinetics upon receptor stimulation (Hofmann et al. 2003) which allows for the passage of monovalent cations, such as sodium and potassium. Due to these kinetics, TRPM5 enters a refractory state quickly after activation. In the refractory state, the ion channel does not respond to activating signals, such as voltage modulation. Therefore, in the refractory state, the TRPM5 ion channel is considered to be desensitized. Through proteolytic studies of other non-TRPM5 voltage-gated Na+ and K+ channels, it was concluded that desensitization results from an intrinsic conformational change within the ion channel. This conformational change is considered to be under the control of a channel subunit or region that is separate from the ion channel region which controls activation. (reviewed in E. R. Kandel, 2000, Principles of Neural Science, McGraw-Hill, New York)
The region controlling inactivation and therefore desensitization of ion channel protein regions have been identified through a number of studies employing techniques such as limited chemical fixation and mutagenesis. In the case of the ligand-gated alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, it was determined through crystallographic and biochemical studies that certain amino acid substitutions produced non-desensitizing AMPA receptors. The L483Y amino acid substitution attenuates desensitization while strengthening the receptor dimer interface through additional contacts made within the ligand binding core. Similarly, cyclothiazide, an allosteric modulator, was shown to attenuate desensitization of the AMPA receptor by binding to a site formed at a dimer interface acting as a cross-linker stabilizing dimer assembly (See e.g., Mayer, M. L. and Horning, M. S. Neuron 42:379-388 (2004); Stern-Bach, Y. et al., Neuron 13:1345-1357 (1994); Sun, Y. et al., Nature 417:245-253 (2002).) In the case of ligand-gated glutamate receptor GluR2, a single amino acid change in the ion channel polypeptide sequence rendered the ion channel non-desensitizing. A highly-conserved tyrosine residue in GluR2 was observed to be necessary for rapid desensitization. When this tyrosine residue was replaced with a tryptophan amino acid, desensitization was attenuated. (See, e.g., Holm, M. M. et al., J. Biol. Chem. 280(42):35469-76 (2005).) The kainate receptors also demonstrate non-desensitization when the binding domain of dimers is stabilized through the generation of intramolecular disulfide bonds. (See, e.g., Priel, A. et al., Neuron 52:1037-1046 (2006).)
One method for testing ion channel activity is to measure changes in cell membrane potential using the patch-clamp technique. (Hamill et al., Nature 294:462-4 (1981)). In this technique, a cell is attached to an electrode containing a micropipette tip which directly measures the electrical conditions of the cell. This allows detailed biophysical characterization of changes in membrane potential in response to various stimuli. Thus, the patch-clamp technique can be used as a screening tool to identify compounds that modulate activity of ion channels. This technique is usually time consuming and normally only allows for fewer than two or three compounds per day to be screened for activity.
Methods of screening test compounds can also be high throughput (i.e., allow for many compounds to be screened quickly), automated, easy to use, sensitive, and selective. Screening assays should also provide a high signal to background noise ratio. (Baxter et al., J. Biomol. Screen. 7:79-85 (2002)). Background noise is the minimal stimulation that a compound produces regardless of its effect on the ion channel. The high ratio makes visualization of positive or negative modulators simpler because the smallest response will be seen over the background measurements. This leads to a clear identification of modulating compounds.
Therefore, there exists a need in the art for a non-desensitizing TRPM5 ion channel that can deactivate slowly such that HTS assays can distinguish compounds that modulate taste by specifically activating TRPM5. A slowly deactivating ion channel is also useful because more compounds can be tested before the channel closes.
The claimed invention provides compositions and methods for HTS assays using a non-desensitizing TRPM5 ion channel.